Non-Viral Delivery of Compounds to Mitochondria

ABSTRACT

A conjugate comprises: (a) a mitochondrial membrane-permeant peptide; (b) an active agent or compound of interest such as a detectable group or mitochondrial protein or peptide; and (c) a mitochondrial targeting sequence linking said mitochondrial membrane-permeant peptide and said active mitochondrial protein or peptide. The targeting sequence is one which is cleaved within the mitochondrial matrix, and not cleaved within the cellular cytoplasm, of a target cell into which said compound is delivered. Methods of use of such compounds are also described.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/514,892 filed Oct. 24, 2003, the disclosure of which isincorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under grant numbersRO1 DK55765 and RO1 DK67763 from the National Institutes of Health. TheU.S. Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns methods and compounds for the delivery ofcompounds, including detectable compounds, proteins and peptides, tomitochondria. The methods and compounds are useful for diagnosticpurposes and for the treatment of mitochondrial diseases such asFriedreich's ataxia, human mitochondrial trifunctional proteindeficiency, and Leber's Hereditary Optic Neuropathy.

BACKGROUND OF THE INVENTION

Mitochondria produce virtually all of the energy supply in tissues withhigh energy demands, and many diseases of impaired mitochondrialfunction have been described involving both mitochondrial and nucleargenomes (B. Robinson, Journal of Bioenergetics & Biomembranes 26:311-316(1994); D. Wallace, American Journal of Human Genetics 57:201-223(1995)). Of the hundreds of proteins that are found within mitochondria,the mitochondrial genome encodes only 13 of these and the rest must beimported from the cytosol (R. Jansen, Human Reproduction 15 Suppl 2:1-10(2000); M. Douglas, and M. Takeda, Trends in Biochemical Sciences10:192-194 (1985)). Based on in vitro observations, proteins targeted tothe mitochondria are thought to be completely synthesized in thecytoplasm and cross the mitochondrial membranes post-translationally (G.Schatz, and B. Dobberstein, Science 271:1519-1526 (1996)). Proteins thatare nuclearly encoded and targeted to the inner and outer membranes,inter-membrane space, or matrix, are aided by the use of presequences atthe N-terminus of the precursor protein (G. Schatz, Journal ofBiological Chemistry 271:31763-31766 (1996); A. Mayer et al. Cell80:127-137 (1995)). Most of these mitochondrial targeting sequences(MTS) consist of 10 to 70 amino acids that are removed by 1 or 2proteolytic steps once inside the mitochondria. At the outermitochondrial membrane the MTS is recognized by a receptor complex(OM37, OM70, or an OM37/OM70 complex), the presequence is passed toOM20, or an OM20/OM22 complex, and membrane translocation proceedsthrough the channel in the outer membrane translocation machinery (TOMcomplex) (T. Komiya, and K. Mihara, Journal of Biological Chemistry271:22105-22110 (1996)). After translocation of the presequence acrossthe outer membrane, a portion of the presequence is recognized by areceptor of the inner membrane (IM) translocation machinery (TIMcomplex) (M. Bauer, et al., Cell 87:33-41 (1996)). The precursor proteinthen proceeds through both the TOM and TIM complexes in conjunction withmitochondrial HSP70 and Tim44 on the matrix side of the IM. Import isdriven by ATP hydrolysis of the HSP70 motor, and the transit peptide iscleaved by the mitochondrial processing peptidase (G. Isaya, et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 89:8317-8321 (1992); T. Omura, Journal of Biochemistry123:1010-1016 (1998)) Finally, the protein is deposited in the matrix orintegrated into the IM (J. Berthold, et al., Cell 81:1085-1093 (1995);C. Ungermann, et al., EMBO Journal 15:735-744 (1996)).

Both nuclear-encoded and mitochondrial-encoded proteins can be mutated,deleted, or be insufficient in amount, leading to functional problems(D. Wallace, Scientific American 277:40-47 (1997); C. Graff, et al.,Journal of Internal Medicine 246:11-23 (1999)). Furthermore, there isnow a growing body of information on how the compartmentation ofmitochondrial proteins, and their function, can be disturbed by acquiredconditions, such as aging, oxidative stress, and ischemia, and which maylead to disease or decreased tissue function (J. Rosenblum, et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 93:4471-4473 (1996); G. Davey, et al., Journal of BiologicalChemistry 273:12753-12757 (1998)). One possible way to alleviate theseproblems is to deliver exogenous protein to mitochondria to replace thedefective or deficient proteins. To date this has been very difficultwith viral or nonviral vectors (R. Owen and T. Flotte, Antioxidants &Redox Signaling 3:451-460 (2001); Y. Bai, et al., Journal of BiologicalChemistry 276:38808-38813 (2001); K. Nakada, et al., Nature Medicine7:934-940 (2001); V. Weissig, and V. Torchilin, Advanced Drug DeliveryReviews 49:127-149 (2001); B. Seo, et al., Proceedings of the NationalAcademy of Sciences of the United States of America 95:9167-9171(1998)). In particular, viral-mediated gene transfer has been associatedwith poor transfection rates and the risk of death in patients (P.Noguchi, N. Engl. J. Med. 348:193-194 (2003). Non-viral techniques, suchas liposome mediated gene transfer, have been even more disappointingwhen applied to vertebrate tissues.

Mitochondria are organelles that are vulnerable to damage for at leastthree reasons. First, mitochondria contain a limited genome,approximately 16 kb in humans. Thus, the majority of proteins essentialfor continued function must be imported into mitochondria. Defects inthese nuclear encoded proteins result in human disease. Furthermore,diseases involving mitochondrial-encoded DNA present a special challengefor potential gene therapy because most of the proteins encoded arehydrophobic and lack a transit peptide. Thus, these proteins aredifficult to keep in the unfolded conformation needed for import. Thismeans their ability to respond appropriately in synthesizing newproteins after damaging conditions such as birth asphyxia, heart attack,stroke, or aging, is limited. Second, mitochondria lack histones andthus, do not have efficient mechanisms for protection and repair of DNAdamage. Consequently, mutations within the mitochondrial DNA (mtDNA) arecumulative and result in disease, such as Leber's Hereditary OpticNeuropathy. Third, mitochondria contain a highly oxidative environmentand generate 95% of the total superoxide radicals in the cell (A.Boveris, Methods in Enzymology 105:429-435 (1984)). Thus, oxidativedamage to the mtDNA and proteins is constant and certain. Mitochondriahave evolved protective mechanisms against this damage, such asmitochondrial superoxide dismutase; however, these can be overwhelmed byabnormal physiology with resultant overproduction of superoxide anddamaging free radicals.

Given that many human diseases involve mitochondrial dysfunction, andthere are currently no satisfactory methods to correct these defects,there is a need to develop techniques to screen for these defects andtherapies to correct mitochondrial defects.

Background on Protein Transduction Domains (PTD). Delivery of drugs andtherapeutic compounds is primarily limited by their ability to penetratethe cell membrane. The bioavailability of compounds targeted tointracellular sites depends on the conflicting requirements of beingsufficiently polar for administration and distribution, yet non-polarenough to diffuse through the non-polar lipid bilayer of the cell (D.Begley, Journal of Pharmacy & Pharmacology 48:136-146 (1996)). Inaddition, the molecular weight of most drugs that can easily traversethe lipid membrane is approximately 500 Da (V. Levin, Journal ofMedicinal Chemistry 23:682-684 (1980)). Thus, most successful compoundshave narrow physical characteristics. Many promising drugs fail becausethey fall outside of this range and efforts to make them available maybe toxic. In addition to this, many sites of action for presumedtherapeutic compounds, such as enzymes or regulatory proteins, requireprocessing and targeting of the compound once inside the cell.

To address these problems, delivery of a gene product into cells hasbeen heavily investigated using both viral and non-viral vectors, aswell as naked DNA and liposome-mediated gene transfer (V. Geromel, etal., Antisense & Nucleic Acid Drug Development 11:175-180 (2001); S.Francis, et al., American Journal of PharmacoGenomics 1:55-66 (2001); B.Cao, et al., Microscopy Research & Technique 58:45-51 (2002)). However,drawbacks with current ‘gene therapies’, such as viral toxicity andinefficient transfection rates, the immune response to viral vectors,and difficulty in creating the gene vector, have limited theirusefulness in gene therapy (M. Rebolledo, et al., Circulation Research83:738-74 (1998); T. Ritter, et al., Biodrugs 16:3-10 (2002)).Furthermore, localizing a gene product within the cell has beendifficult. For example, attempts to deliver proteins to mitochondriawithin the cells to correct defects in their function have been limited(P. Seibel, et al., Nucleic Acids Research 23:10-17 (1995)). The use ofmitochondrial targeting sequences to localize fusion proteins withinmitochondria is not a new concept. Fusion proteins made withmitochondrial signal sequences have been transfected into cultured cellsand shown to not only be targeted to mitochondria, but also processed,allowing for complete localization and functionality of the fusedprotein (C. Zhang, et al., Biochemical & Biophysical ResearchCommunications 242:390-395 (1998); B. Seaton, and L. Vickery, Archivesof Biochemistry & Biophysics 294:603-608 (1992)). However, the transferof this technology to tissues in vivo has not been successful, in part,because of problems with delivery of the gene product.

Recently, a novel strategy for delivery of synthetic compounds has beendescribed and is being actively investigated by both industry andacademic researchers (R. Service, Science 288:28-29 (2000)). Positivelycharged, cationic peptides, are known to cross cell membranesindependent of receptors or specific transport mechanisms (S. Schwarze,et al., Science 285:1569-1572 (1999); A. Ho, et al., Cancer Research61:474-477 (2001); M. Morris, et al., Nature Biotechnology 19:1173-1176(2001); M. Pooga, et al., FASEB Journal 12:67-77 (1998); D. Derossi, etal., Journal of Biological Chemistry 271:18188-18193 (1996); G.Pietersz, et al., Vaccine 19:1397-1405 (2001); G. Elliott, and P.O'Hare, Cell 88:223-233 (1997); W. Derer, et al., FASEB Journal16:132-133 (2002); E. Will, et al., Nucleic Acids Research 30:e59(2002); J. Rothbard, et al., Journal of Medicinal Chemistry 45:3612-3618(2002); L. Chen, et al., Chemistry & Biology 8:1123-1129 (2001); P.Wender, et al., Proceedings of the National Academy of Sciences of theUnited States of America 97:13003-13008 (2000)). The transport involvesprotein transduction domains (PTD) that are highly charged, shortpeptides (˜10 to 20 amino acids), containing basic amino acids(arginines and lysines), and that have the ability to form hydrogenbonds. The ability of PTD's to cross cell membranes is alsoconcentration-dependent.

Multiple investigators have found that attachment of nucleic acids,peptides, and even large proteins to these PTD's will allow theirtransduction across all cell membranes in a highly efficient manner (S.Schwarze and S. Dowdy, Trends in Pharmacological Sciences 21:45-48(2000)). Three PTD's have been described which share the commoncharacteristics of being potential DNA binding proteins: HIV-TAT, VP22,and Antennapedia (S. Schwarze, et al., Science 285:1569-1572 (1999); D.Derossi, et al., Journal of Biological Chemistry 271:18188-18193 (1996);G. Elliott, and P. O'Hare, Cell 88:223-233 (1997)). Based on computermodeling and the prediction that these PTD's often have a strongα-helical character with a face of basically charged residues(arginines), investigators have begun to create synthetic peptides withgreater ability to efficiently and quickly transduce across cellmembranes. The exact mechanism of protein transduction is not known butis not receptor mediated and is independent of temperature making itunlikely that endocytosis or transporter mechanisms are involved (D.Mann and A. Frankel, EMBO Journal 10:1733-1739 (1991)). Furthermore,treatment of cells with drugs that inhibit cellular transport, such asbrefeldin A (inhibits golgi transport), do not affect transduction ofPTD's (G. Elliott, and P. O'Hare, Cell 88:223-233 (1997)).

Recently it was shown that the PTD derived from the HIV genome, HIV-TAT(trans-activator of transcription, “TAT”), has the ability to moveattached peptides, large proteins, and nucleic acids across virtuallyall cell membranes, including brain, in a non-receptor mediated fashion(S. Schwarze, et al., Science 285:1569-1572 (1999); G. Cao, et al.,Journal of Neuroscience 22:5423-5431 (2002); A. Gustafsson, et al.,Circulation 106:735-739 (2002); H. Nagahara, et al., Nature Medicine4:1449-1452 (1998)). The attached proteins are refolded into an activeconformation once inside the cell and are biologically active. The fulllength TAT protein, originally described in 1988, by Green andLowenstein, and is an 86 amino acid protein encoded by the HIV virus (S.Fawell, et al., Proc. Natl. Acad. Sci. U.S.A. 91:664-668 (1994); A.Frankel, and C. Pabo, Cell 55:1189-1193 (1988); M. Green and P.Loewenstein, Cell 55:1179-1188 (1988)). More specifically, an 11 aminoacid arginine- and lysine-rich portion of the TAT sequence, YGRKKRRQRRR,conjugated to peptides that do not normally cross membranes, is able totransduce across cell membranes and deliver a biologically active fusionprotein to tissues. Furthermore, when a TAT-fusion protein was injectedinto mice for two weeks, there were no gross signs of neurologicalproblems or system distress. Previously, TAT-fusion proteins were shownto be capable of delivering an active fusion protein that affectsmitochondrial function, though in both cases, the fusion protein was notprocessed by the mitochondria. (G. Cao, et al., Journal of Neuroscience22:5423-5431 (2002); A. Gustafsson, et al., Circulation 106:735-739(2002)).

In summary, PTD's appear to offer a method for the efficient and rapidtransport of highly charged, polar compounds across virtually all cellmembranes and tissues in a concentration-dependent manner. This includesthe mitochondrial membranes. These PTD's are well tolerated with onlyminimal detrimental effects seen at high concentrations in cell culture.However, because PTD-fusion proteins follow a concentration gradient,their use as therapeutic vehicles is limited by loss of the PTD-fusionprotein from the cell unless the protein is bound or processed.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a compound or conjugatecomprising: (a) a mitochondrial membrane-permeant peptide; (b) an activeagent or compound of interest such as a mitochondrial protein orpeptide, nucleic acid, drug, or signaling agent; and (c) a mitochondrialtargeting sequence linking said mitochondrial membrane-permeant peptideand said active mitochondrial protein or peptide. The targeting sequenceis one which is cleaved within the mitochondrial matrix, and not cleavedwithin the cellular cytoplasm, of a target cell into which said compoundis delivered.

A second aspect of the present invention is a composition comprising acompound or conjugate as described above in a pharmaceuticallyacceptable carrier.

A third aspect of the present invention is a method of delivering acompound of interest to the mitochondria of a cell comprising contactinga compound or conjugate as described above to a cell, or a tissuecontaining said cell, in vitro or in vivo so that said compound ofinterested is delivered into the mitochondria of said cell.

A further aspect of the present invention is a method of treating amitochondrial disorder in a subject in need thereof, comprisingadministering a compound or conjugate as described above to said subjectin an amount effective to treat said mitochondrial disorder.

A further aspect of the present invention is the use of a compound orconjugate as described above for the preparation of a composition ormedicament for carrying out a method as described above.

The invention is explained in greater detail in the drawings herein andthe specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: β-Oxidation Spiral. The 3 enzymatic steps of the TFP are shownwith blocks (red) occurring at 3-hydroxyacyl-CoA dehydrogenase (LCHAD)and before the hydratase reaction (complete TFP deficiency).

FIG. 2: Predicted sizes of precursor (A), intermediate (B) and mature(C) forms of TAT-mMDH-eGFP after incubation with mitochondria matrixcontents (D). Panel (E): Protection of TAT-mMDH-eGFP, lanes 1 (control)and 2 (transduced), and TAT-GFP, lanes 3 (control) and 4 (transduced)from protease digestion in intact mitochondria.

FIG. 3: TAT-mMDH-eGFP, but not TAT-GFP, co-localizes to mitochondria.NIH 3T3 or differentiated PC12 cells, were incubated with 0.01 mg/ml ofeither TAT-mMDH-eGFP or TAT-GFP for 1 hour, and imaged at later times(left side of rows). Cells were then stained with CMX-Rosamine prior toanalysis by confocal microscopy. The localization of GFP alone (green,column A) or CMX-Rosamine alone (red, column B) and co-staining (yellow,column C) is shown. Yellow stain indicates co-localization of GFP andCMX-Rosamine within mitochondria. Cell line is indicated on the rightfor each row. Micron bars indicating magnification are 10 microns and 5microns in length for NIH 3T3 and PC12 cells, respectively, and allsamples represent an N of 2.

FIG. 4: Kinetics of TAT fusion protein transduction. TAT-GFP transducesinto PC12 cells with pseudo-first order kinetics. Cells were incubatedwith 0.01 mg/ml TAT-GFP for progressive times, images taken with theconfocal microscope, and the average fluorescence minus backgroundanalyzed. The average relative fluorescence at each time point isplotted on the y axis. All points are the average of seven image fields.The solid line was obtained by fitting to an equation,F=F_(max)[1−e^(−t/t1/2)], which yielded F_(max)=38.8±2.0 andt_(1/2)=13.2±1.9 min. The error bars represent the standard deviation.

FIG. 5: Mouse tissues after TAT-fusion protein. SVEV mice were injectedip with 2 mg/kg TAT-GFP or TAT-mMDH-eGFP and sacrificed 17 hours, or 5days later. All comparable samples were scanned with the same pinholeand detector gain settings. A,B,C: TAT-mMDH-eGFP 17 hr after injection.D,E,F: TAT-GFP 17 hr after injection. G,H,I: TAT-mMDH-eGFP 5 d afterinjection. J,K,L: TAT-GFP 5 d after injection.

FIG. 6: Intracellular location of TAT-mMDH-eGFP and TAT-GFP 5 days afterinjection into mice. (A) 5 days post injection of TAT-mMDH-eGFP orTAT-GFP into SVEV mice, mitochondria (lanes 1 and 5), cytosolic (lanes 3and 6), and mitochondrial wash (lane 4) fractions were isolated fromliver and resolved along with purified TAT-mMDH-eGFP (lane 2) andTAT-GFP (lane 7) via SDS-PAGE. Proteins were transferred to nylonmembranes and probed with antibodies against GFP. As controls, cytosolicand mitochondrial fractions were also probed with antibodies againstcytochrome c oxidase subunit vb (B) and cathepsin L (C). Data arerepresentative of results from two independent experiments.Cyto=cytosolic fraction and Mito=mitochondrial fraction.

FIG. 7: Placental transfer. Pregnant mice were injected ip with 2 mg/kgTAT-mMDH-eGFP at 18 d gestation and sacrificed 24 and 48 hr later. A)Maternal heart 24 hr post injection. B) Fetal heart 24 hr post maternalinjection. C) Newborn pup heart 48 hr post maternal injection. Notearrows pointing to RBC's. D) Adult kidney 5 d post ip injectionTAT-mMDH-eGFP. E) Adult kidney 5 post ip injection TAT-GFP.

FIG. 8: Construction of TAT-mMDH-eGFP Fusion Protein. The pTAT-HA vectorcontains a multiple cloning site (MCS) flanked by a termination codon(Term) and hemagglutinin (HA) tag for antibody detection. The sequenceof the TAT peptide is displayed in red and is preceded by a His tag fornickel affinity purification. The cDNA for enhanced Green FluorescentProtein (eGFP) was subcloned into the NCO I restriction site in framewith the mMDH transit peptide, expressed in E. coli, purified usingnickel affinity chromatography, and denatured with urea and ion exchangechromatography prior to use.

FIG. 9: PET Scan of mouse heart. An adult mouse was injected with 1 mCi[¹⁸F]2-fluoro-2-deoxy-D-glucose (FDG). Images were taken 1 hour aftertail vein injection. Static images taken over 20 min show very gooddetail of cardiac uptake of labeled glucose. Images gated for heart ratebased on the electrocardiogram, and crosshairs are centered on theheart. This figure shows the feasibility and resolution of the MicroPETfor imaging the mouse. Right (R), Left (L), Dorsal (D), Ventral (V).

FIG. 10: Construction of TAT-mMDH-eGFP Fusion Protein. The pTAT-HAvector contains a multiple cloning site (MCS) flanked by a terminationcodon (Term) and hemagglutinin (HA) tag for antibody detection. Thesequence of the TAT peptide is displayed in red and is preceded by a Histag for nickel affinity purification. The cDNA for enhanced GreenFluorescent Protein (eGFP) was subcloned into the NCO I restriction sitein frame with the mMDH transit peptide, expressed in E. coli, purifiedusing nickel affinity chromatography, and denatured with urea and ionexchange chromatography prior to use.

FIG. 11: TAT-fusion protein transduction causes phosphatidylserine flip.In FIG. 11, starting with B (A not shown), phosphatidylserine (PS) flipis detected by Annexin-V staining in cell culture. Cells were treatedfor 12 hours with 300 μM H₂O₂ and stained with Annexin-V (B) As acontrol untreated cells were also stained with Annexin-V (C). Cells wereincubated with 0.01 mg.ml TAT-GFP and stained with Annexin V (D,E).Cells were also pretreated with polylysine prior to incubation withTAT-GFP and Annexin-V staining (F) Fluorescent levels of activatedCaspase3 were measured in cultured cells that were treated with either0.01 mg/ml (29.5 μM) TAT-GFP (diagonal bars) or hydrogen peroxide for 12hours (solid black bars) (G)

FIG. 12. Transduction of human Frataxin into NIH 3T3 cells. Analysis byconfocal microscopy shows the TAT-Frataxin fusion protein transducesinto NIH-3T3 cells. Cells were stained with CMX-Rosamine alone (A). ADIC image shows cell structure (B). Cells were incubated with humanFrataxin fusion protein and labeled with fluorescene (bright greencolor) (C). Staining shows co-localization of CMX-Rosamine and humanFrataxin within mitochondria (D).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The disclosures of all United States Patent references cited herein areto be incorporated by reference herein in their entirety.

Compounds or conjugates of the present invention are prepared from threecomponents: (a) a mitochondrial membrane-permeant peptide; (b) acompound of interest such as a detectable group or compound, an activemitochondrial protein or peptide, nucleic acids, drug or signalingagent; and (c) a mitochondrial targeting sequence.

Mitochondrial membrane-permeant peptides that may be used to carry outthe present invention. While any suitable peptide may be used, preferredpeptides are HIV-TAT peptides. For example, the Tat peptide can compriseany sequential residues of the Tat protein basic peptide motif 37-72(Vives et al. 1997) (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72 (SEQ IDNO:1). The minimum number of amino acid residues can be in the range offrom about three to about six, preferably from about three to aboutfive, and most preferably about four, i.e., the minimal requirement forone alpha helical turn. One embodiment comprises Tat protein residues48-57 (GRKKRRQRRR) (SEQ ID NO:2). See, e.g., U.S. Pat. No. 6,348,185 toPiwnica-Worms. Additional mitochondrial permeant peptide transductiondomains comprise the sequences listed in Table 1.

TABLE I Protein Transduction Domains PTD Name Sequence Notes Ref. TATYGRKKRRQRRR HIV DNA binding S. Schwarze, et al., Science 285: 1569- (SEQID NO: 3) domain 1572 (1999). PTD-4 YARAAARQARA 33× more efficient A.Ho, et al., Cancer Research 61: 474- (SEQ ID NO: 4) than TAT 477 (2001).Pep-1 KETWWETWWTEWS 3 domains. Does not M. Morris, et al., NatureQPKKKRKV (SEQ ID require cross-linking Biotechnology 19: 1173-1176(2001). NO: 5) to transduced protein Transportan GWTLNSAGYLLGKINNeuropeptide of M. Pooga, et al., FASEB Journal LKALAALAKKIL galanin(1-13) and 12: 67-77 (1998). (SEQ ID NO: 6) mastoparan (14-27)Antennapedia RQIKIWFQNRRMKW DNA binding D. Derossi, et al., Journal ofBiological KK (SEQ ID NO: 7) domain Chemistry 271: 18188-18193 (1996);G. Pietersz, et al., Vaccine 19: 1397- 1405 (2001). VP22 DAATATRGRSAASRPHSV-1 tegument G. Elliott, and P. O'Hare, Cell 88: 223- TERPRAPARSASRPRprotein 233(1997); W. Derer, et al., FASEB RPVE (SEQ ID NO: 8) Journal16: 132-133 (2002). Cre Cre Recombinase, ~41 DNA binding E. Will, etal., Nucleic Acids Research kDa. protein from Cre 30(12): e59 (2002)recombinase R₇, R₉, r₉ Oligomers of D- and L- D-Arg (r₉) 100-fold > J.Rothbard, et al., Journal of arginine TAT uptake rate MedicinalChemistry 45: 3612-3618 (2002); L. Chen, et al., Chemistry & Biology 8:1123-1129 (2001); P. Wender, et al., Proceedings of the National Academyof Sciences of the United States of America 97: 13003- 13008 (2000)

Compounds of interest that may be used to carry out the presentinvention are proteins or peptides that have activity in themitochondrial for any purpose, including diagnostic, therapeutic, andhistological purposes. In one embodiment of the invention, the activemitochondrial protein or peptide is Frataxin; in another embodiment, themitochondrial protein or peptide is trifunctional protein alpha. Theactive mitochondrial proteins or peptides are preferably mammalian inorigin, and most preferably human in origin.

Mitochondrial targeting sequences that may be used to carry out thepresent invention include any sequence that is cleaved within themitochondrial matrix, but not cleaved within the cellular cytoplasm, ofa target cell into which the compound is delivered. Particular examplesinclude the mitochondrial malate dehydrogenase cleavage sequence andmitochondrial creatine kinase (MtCK) sequence. The targeting sequencemay be one that is not ordinarily found associated with the activeprotein or peptide (that is, a heterologous sequence), or may be onethat is native to the active protein or peptide as found in the Frataxinand tri-functional proteins or peptides (TFP) (that is, a homologoussequence).

Compounds of the present invention can be prepared and formulated fromthe above in accordance with techniques known in the art, including butnot limited to those described in U.S. Pat. No. 6,348,185 toPiwnica-Worms.

Subjects to be treated by the compounds and methods described hereininclude both human subjects and animal subjects (particularly mammaliansubjects such as mice, cats, dogs, etc.) for medical, veterinary, anddrug development purposes. The subject, including human subjects, may bemale or female, and may be at any stage of development, includingneonate, infant, child, adolescent, adult and geriatric subjects. Cellsor tissues used to carry out the present invention include those fromlike subjects, such as muscle, nerve, and liver cells and tissues.

Compounds for conjugation with mitochondrial membrane-permeant peptides.An active compound, active agent or compound of interest of the presentinvention may be any suitable compound and includes, but is not limitedto, peptides, proteins, enzymes (both protein and non-protein), nucleicacids, oligonucleotides, lipids, phospholipids, steroids, metalchelators, free radical scavengers, vitamins, drugs and prodrugs. Theactive agent may be active for any purpose and thus may be a therapeuticagent, a diagnostic agent, an imaging/staining agent, etc.

In some embodiments the active agent is a detectable group, e.g., afluorescent group such as green fluorescent protein, a luminescentgroup, a spin label, aphotosensitizer group for singlet oxygengeneration, a photocleavable moiety, a chelating center, a heavy atom, aradioactive isotope, an isotope detectable by nuclear magneticresonance, a paramagnetic atom (see, e.g., U.S. Pat. No. 6,686,458; U.S.Pat. No. 6,294,340; U.S. Pat. No. 6,251,584, etc.).

Conjugates. The mitochondrial-permeant peptides of this invention can beconjugated to an array of active compounds as described above.Conjugation may be direct or indirect (e.g., through an intermediatemolecule or binding pair) and may be covalent or noncovalent. Theconjugations can be carried out using any suitable method, including butnot limited to those described below.

Peptides/proteins conjugated to other peptides/proteins. If bothcomponents are peptides or proteins then the conjugation may be carriedout by recombinant methods [EP388 758 (referenced by Curiel et al inU.S. Pat. No. 6,274,322); Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed. 1989)); U.S. Pat. No. 6,803,053].

Peptides/proteins conjugated to nucleic acids. (1) The conjugation ofpeptides or proteins to nucleic acids can be done using the methods ofCuriel et al. in which substances having an affinity for nucleic acidsare bound to the proteins or peptides, (U.S. Pat. No. 5,521,291; U.S.Pat. No. 6,274,322; [also U.S. Pat. No. 6,077,663; U.S. Pat. No.5,354,844; U.S. Pat. No. 5,792,645; U.S. Pat. No. 5,547,932; U.S. Pat.No. 5,981,273; U.S. Pat. No. 6,022,735; the disclosures of which arefully incorporated by reference herein). Substances with an affinity fornucleic acid which may be used include, for example, homologouspolycations such as polylysine, polyarginine, polyornithine orheterologous polycations having two or more different positively chargedamino acids, these polycations possibly having different chain lengths,and also non-peptidic synthetic polycations such as polyethyleneimine.Other substances with an affinity for nucleic acid which are suitableare natural DNA-binding proteins of a polycationic nature such ashistones or protamines or analogues or fragments thereof.

The coupling of the peptide or protein with the polycation may becarried out by means of disulphide bridges, which can be cleaved againunder reducing conditions (e.g. when usingsuccinimidyl-pyridyldithiopropionate; by means of compounds which aresubstantially stable under biological conditions (e.g. thioethers byreacting maleimido linkers with sulfhydryl groups of the linker bound tothe second component) or by bridges which are unstable under biologicalconditions, e.g. ester bonds, or acetal or ketal bonds which areunstable under slightly acidic conditions.

Furthermore, the binding of peptides or proteins to a substance havingan affinity for nucleic acids such as polyamine can be carried out bymeans of transglutaminase. Transglutaminases comprise a number ofdifferent enzymes which catalyze the formation ofepsilon-(gamma-glutamyl)lysine bonds in the presence of Ca⁺⁺ and withcleaving of NH₃.

Additional methods of preparing the polycation conjugates include:coupling the peptide or protein to the polycation via a biotin-proteinbridge using streptavidin or avidin. The streptavidin or avidin is thenchemically conjugated to polylysine in a similar manner to the productof transferrin-polylysine conjugates. Binding between proteins andpolylysine may also be achieved by coupling polylysine with a lectinwhich has an affinity for a glycoprotein, the bonding in such aconjugate being effected by means of the bond between the lectin and theglycoproteins.

(2) A further method of binding target molecules to nucleic acids iscalled Systematic Evolution of Ligands by Exponential Enrichment, termedSELEX, is described in U.S. Pat. No. 5,475,096 entitled “Nucleic AcidLigands,” and U.S. Pat. No. 5,270,163 entitled “Methods for IdentifyingNucleic Acid Ligands,” both of which are specifically incorporated byreference herein. The SELEX process provides a class of productsreferred to as nucleic acid ligands, each ligand having a uniquesequence and property of binding specifically to a desired targetcompound or molecule. The SELEX method includes steps of: (1) contactingthe mixture with the target under conditions favorable for binding, (2)partitioning unbound nucleic acids from those nucleic acids which havebound specifically to target molecules, (3) dissociating the nucleicacid-target complexes, (4) amplifying the nucleic acids dissociated fromthe nucleic acid-target complexes to yield a ligand-enriched mixture ofnucleic acids, (5) then reiterating the steps of binding, partitioning,dissociating and amplifying through as many cycles as desired to yieldhighly specific high affinity nucleic acid ligands to the targetmolecule. U.S. Pat. No. 6,737,236

Furthermore, the basic SELEX method has been modified. For example,SELEX based methods for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking atarget molecule, including a protein or peptide, have been described(U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, U.S. Pat. No.6,730,482). Methods that modify nucleotides and confer improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics including chemical substitutions atthe ribose and/or phosphate and/or base positions, are also described(U.S. Pat. No. 5,660,985, U.S. patent application Ser. No. 09/134,028,d, U.S. patent application Ser. No. 08/264,029).

A method for the synthesis of oligodeoxynucleotides terminated by5′-amino-5′-deoxythymidine has been described (Bruick et al. (1997)Nucleic Acids Res. 25:1309-1310. This method uses a DNA template todirect the ligation of a peptide to an oligonucleotide, in which thepeptide is presented by a second oligonucleotide in the foam of areactive thioester-linked intermediate.

Peptides/proteins conjugated to other macromolecules. (1) Cycloadditionreactions, including Diehls-Alder reactions, 1,3-dipolar cycloadditionreactions and [2+2] cycloaddition reactions, as described in U.S. Pat.No. 6,737,236; which is specifically incorporated by reference herein,provide a chemoselective and highly efficient method for derivatizing orconjugating macromolecules with other molecular entities. This methoddescribes conjugation of peptides with such molecular entities asoligonucleotides, nucleic acids, proteins, peptides carbohydrates,polysaccharides, glycoproteins, lipids, hormones, drugs or prodrugs.Accordingly, a macromolecule bearing a moiety capable of undergoing acycloaddition reaction, is reacted with another molecular entity bearinga moiety capable of undergoing a cycloaddition reaction with the moietyattached to the macromolecule to yield via a cycloaddition reactionefficient conjugation of the molecular entity to the macromolecule. Morespecifically, a macromolecule bearing either a diene or dienophilemoiety is reacted with another molecular entity bearing either adienophile or a diene moiety, respectively, to yield via a cycloadditionreaction efficient conjugation of the molecular entity to themacromolecule. The Diels-Alder reaction, in particular, is an idealmethod for covalently linking large water soluble macromolecules withother compounds as the reaction rate is accelerated in water and can berun at neutral pH. (Rideout and Breslow (1980) J. Am. Chem. Soc.102:7816). Additionally, the nature of the reaction allowspost-synthetic modification of the hydrophilic macromolecule withoutexcess reagent or hydrolysis of the reagent (U.S. Pat. No. 6,737,236).

The method of cycloaddition reactions can be extended to the conjugationof any macromolecule with another molecular entity with themacromolecule or molecular entity including but not limited tooligonucleotides, nucleic acids, proteins, peptides carbohydrates,polysaccharides, glycoproteins, lipids, hormones, drugs or prodrugs. Forexample, a peptide or protein that contains an amino acid building blockwhich has been derivatized with a diene or dienophile, such as0-3,5-hexadiene-tyrosine or serine, or N-maleimidolysine, can beconjugated to another molecular entity including, but not limited to,another peptide, an oligonucleotide, nucleic acid, carbohydrate,detector molecule etc. without limitation. Natural macromolecules suchas proteins can be derivatized with a diene or dienophile bearingheterobifunctional crosslinking reagent, such as the NHS ester of3-(4-maleimidophenyl)-propionic acid, which allows subsequentconjugation to a macromolecule or diagnostic detector molecule bearing acorresponding diene or dienophile group, (U.S. Pat. No. 6,737,236).

(2) Finally, bioconjugates can be made according to the method of U.S.Pat. No. 6,790,827, entitled “Bioconjugation of Macromolecules,” whichdescribes the method of creating bioconjugates between a bioactive agentand an organocobalt complex and is specifically incorporated byreference herein. In this method the bioactive agent is covalentlybonded directly or indirectly (via a spacer—polymethylene, ester,carbonate, ether, acetal or any combination of two or more of theseunits) to the cobalt atom of the organocobalt complex. The organocobaltcomplex binds the bioactive agent covalently to the cobalt such that thecobalt-bioactive agent bond is readily cleavable.

For this method the bioactive agent is any biologically active moleculethat can form a conjugate with an organocobalt complex. The bioactiveagent includes but is not limited to, peptides, oligopeptides, proteins,apoproteins, glycoproteins, antigens and antibodies or antibodyfragments, protein analogs in which at least one non-peptide linkagereplaces a peptide linkage, enzymes, coenzymes, enzyme inhibitors, aminoacids and their derivatives, hormones, lipids, phospholipids, vitamins,minerals and nutritional additives, nucleotides, oligonucleotides,polynucleotides, and their art-recognized and biologically functionalanalogs and derivatives, plasmids, cosmids, artificial chromosomes, etc.

The organocobalt complex is any organic complex containing a cobalt atomhaving bound thereto 4-5 nitrogen and/or chalcogens such as oxygen,sulfur, etc., as part of a multiple unsaturated heterocyclic ringsystem. Suitable organocobalt complexes include, but are not limited to,cobalamin (coenzyme B₁₂), Co[SALEN] (which is a cobalamin analogue),organo(pyridine)-bis(dimethylglyoximato)cobalt, corrinoids andderivatives or analogues of any of the preceding, as well aspharmaceutically acceptable salts. The organocobalt complexes may beunsubstituted or substituted with conventional organic functional groupsthat will not alter the basic nature of the organocobalt complex, (U.S.Pat. No. 6,790,827).

Substituents which may be found on the organocobalt complex includeamino, nitro, halogen (bromine, chlorine), sulfito, C₂₋₆-alkene and C₂₋₆alkyne. For example, the organocobalt complex can be formed having anitro and/or halo (e.g., bromo) derivative of the corrin ring or havingan extended conjugation with exocyclic olefin or alkylene groups. Otherderivatives include cobalamin lactone, cobalamin lactame and those inwhich the benzimidiazole ring (e.g., of cobalamin, green corrinoid, andthe like) are substituted with e.g., one or more halogen (bromine,chlorine), hydroxy or C₁₋₆ alkyl. Further derivatives include anilide,ethylamide, mono-, di- or tri-carboxylic acid or proprionamidederivatives of cobalamin of Vitamin B₁₂.

Background on Human Mitochondrial Trifunctional Protein Deficiency.Mitochondria primarily utilize two different fuels for the generation ofoxidative energy: pyruvate, derived from cytosolic glycolysis ofglucose, and fatty acids which enter the β-oxidative pathway. Glucose isthe major fuel for brain which cannot utilize fatty acids. It can,however, readily oxidize the ketone bodies derived from acetyl CoA andacetoacetyl CoA produced from β-oxidation in the liver. Fatty acidoxidation is a multi-step pathway that generates more than six times theenergy of an equivalent mass of hydrated glycogen. Thus, fatty acidoxidation is the major source of energy for many tissues such as heartand skeletal muscle, where high density of energy is important. Fuelsources change dramatically around the time of birth in humans. Whileglucose is the predominant fuel for the fetus, after birth, fats derivedfrom milk become more important in metabolism. Thus, defects in fattyacid metabolism frequently manifest themselves in the first year oflife, in part, because of this switch.

Both pyruvate (from glycolysis) and fatty acids are selectivelytransported into the mitochondrial matrix where they are converted toacetyl CoA and enter the tricarboxylic acid cycle (TCA). The conversionof fatty acids to acetyl CoA in the β-oxidation spiral occurs via therepetitive step-wise removal of two carbon units from the carboxyl endof fatty acid molecules producing one molecule of acetyl CoA, and onemolecule each of FADH₂ and NADH (FIG. 1) (D. Kelly, and A. Strauss, NewEngland Journal of Medicine 330:913-919 (1994)). The first reaction ofthe four enzymatic steps in the β-oxidation spiral is catalyzed by theacyl-CoA dehydrogenases, which are a family of enzymes with overlappingsubstrate specificity. These consist of a short chain acyl-CoAdehydrogenase (SCAD), a medium chain acyl-CoA dehydrogenase (MCAD), along chain acyl-CoA dehydrogenase (LCAD) and a very long chain acyl-CoAdehydrogenase (VLCAD). The last three reactions of the fatty acidβ-oxidation spiral for longer chain substrates are catalyzed by themitochondrial trifunctional protein (TFP) which consists of the longchain enoyl-CoA hydratase, the long chain 3-hydroxyacyl-CoAdehydrogenase (LCHAD), and the long chain 3-ketoacyl-CoA thiolase. TheTFP complex is a heterocomplex composed of four α and four β chainsassociated with the inner mitochondrial membrane (Y. Uchida, et al.,Journal of Biological Chemistry 267:1034-1041 (1992)). Thecarboxy-terminal domain of the α-chain (TFPα) contains the LCHADactivity that catalyzes the 3^(rd) step of the β-oxidation spiral. TheN-terminal domain of TFPα contains the long chain 3-enoyl-CoA hydrataseactivity that catalyzes the 2^(nd) step. The β-chain (TFPβ) contains thelong chain 3-ketoacyl-CoA thiolase activity and catalyzes the 4^(th)step in the β-oxidation spiral.

Defects in fatty acid oxidation are among the most common of inheritedrecessive metabolic disorders with an estimated incidence in thepopulation of ˜1 in 12,000 (J. Wood, et al., Pediatrics 108:E19 (2001)).These defects can present with a Reye-like syndrome in childrenincluding encephalopathy, hypoglycemia, and metabolic derangements thatcan lead to death. Of these, defects in medium chain acyl-Coenzyme A(CoA) dehydrogenase are the most common with a prevalence of 1 in 10,000to 1 in 15,000 live births and can have a mortality as high as 25% (K.Tanaka, et al., Human Mutation 1:271-279 (1992); C. Wilson, et al.,Archives of Disease in Childhood 80:459-462 (1999)). Defects may alsooccur in the TFP which represents 3 of the 4 enzymatic steps fordegradation of fatty acids to fuel mitochondria. These defects fall intotwo biochemical subgroups (S. Ushikubo, et al., American Journal ofHuman Genetics 58:979-988, 1996; J. Ibdah, et al., Journal of ClinicalInvestigation 102:1193-1199 (1998); J. Brackett, et al., Journal ofClinical Investigation 95:2076-2082 (1995); S. Jackson, et al., Journalof Clinical Investigation 90:1219-1225 (1992)). The first group hasisolated LCHAD deficiency but normal or slightly reduced hydratase andthiolase activities. The second group has complete TFP deficiency. Inchildren, these present most often as a deficiency in LCHAD. Childrenwith LCHAD deficiency may present at a few months of age with an acutemetabolic crises consisting of low blood sugar (hypoglycemia) andhepatic encephalopathy that can progress rapidly to death. Theseepisodes frequently follow a prolonged period of fasting or stress, suchas might be experienced during the flu with vomiting and diarrhea. Othermanifestations of LCHAD deficiency may include cardiomyopathy, muscleweakness, or sudden infant death syndrome (R. Pollitt, Journal ofInherited Metabolic Disease 18:473-490 (1995)).

An interesting finding has been that most heterozygous women who carriedLCHAD deficient fetuses frequently develop serious, and life-threateningmetabolic disorders such as acute fatty liver of pregnancy (AFLP) or thesyndrome of hemolysis, elevated liver enzymes, and low platelets (HELLPsyndrome) (J. Ibdah, et al., Molecular Genetics & Metabolism 71:182-189(2000)). The development of AFLP is devastating with high neonatal andmaternal morbidity and mortality and recent data strongly suggests thatthe block at LCHAD in the fetus or placenta causes accumulation oflong-chain 3-hydroxyacyl metabolites in the mother that are highly toxicto the liver (J. Ibdah, et al., New England Journal of Medicine340:1723-1731 (1999)). Supporting this hypothesis is the observationthat this effect on the mother is not seen in infants completelydeficient in TFP. The prevalence of AFLP is high at 1 in 7,000 to 1 in13,000 deliveries and expectant mothers will develop hepatic failurewith encephalopathy in the 3^(rd) trimester if not diagnosed anddelivery quickly accomplished (C. Riely, Seminars in Liver Disease7:47-54 (1987); T. Knox, and L. Olans, New England Journal of Medicine335:569-576 (1996); M. Castro, et al., American Journal of Obstetrics &Gynecology 174:211-216 (1996)).

Background on the Mitochondrial Trifunctional Protein Animal Model. Ananimal model of the human TFP deficiency has been generated (J. Ibdah,et al., Journal of Clinical Investigation 107:1403-1409 (2001)).Biochemical and histologic abnormalities of the homozygous animalsclosely approximates that of TFP deficiency in the human (R. Wanders, etal.; Journal of Inherited Metabolic Disease 22:442-487 (1999)).

Pharmaceutically Acceptable Salts of Peptide Complexes. Like aminoacids, peptides and proteins are ampholytes, i.e., they act as bothacids and bases by virtue of the presence of various electron-donor andacceptor moieties within the molecule. The peptide complexes of thepresent invention can therefore be used in the free acid/base form, inthe form of pharmaceutically acceptable salts, or mixtures thereof, asis known in the art. Such salts can be formed, for example, with organicanions, organic cations, halides, alkaline metals, etc.

The term “pharmaceutically acceptable salts” embraces salts commonlyused to form alkali metal salts and addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Suitable pharmaceutically acceptable baseaddition salts of the present peptide complexes include metallic saltsand organic salts.

Preferred metallic salts include, but are not limited to, appropriatealkali metal (group Ia) salts, alkaline earth metal (group IIa) salts,and other physiologically acceptable metals. Such salts can be prepared,for example, from aluminum, calcium, lithium, magnesium, potassium,sodium, and zinc.

Organic salts can be prepared from tertiary amines and quaternaryammonium salts, including in part, tromethamine, diethylamine,N,N′-dibenzyl-ethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methyl-glucamine), and procaine.

Such salts can also be derived from inorganic or organic acids. Thesesalts include but are not limited to the following: acetate, adipate,alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate,cyclopentanepropionate, dodecyl sulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate,nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate.

The basic nitrogen-containing groups can be quaternized with agents suchas lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides, and iodides; dialkyl sulfates such as dimethyl,diethyl, dibuytl, and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkylhalides such as benzyl and phenethyl bromides, and others.

All of these salts can be prepared by conventional means from thecorresponding peptide complex disclosed herein by reacting theappropriate acid or base therewith. Water- or oil-soluble or dispersibleproducts are thereby obtained as desired.

Formulations/Pharmaceutical Compositions. The compounds of the presentinvention can be formulated as pharmaceutical compositions. Suchcompositions can be administered orally, parenterally, by inhalationspray, rectally, intradermally, transdermally, or topically in dosageunit formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. Topicaladministration may also involve the use of transdermal administrationsuch as transdermal patches or iontophoresis devices. The termparenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y. (1980).

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are usefulin the preparation of injectables. Dimethyl acetamide, surfactantsincluding ionic and non-ionic detergents, and polyethylene glycols canbe used. Mixtures of solvents and wetting agents such as those discussedabove are also useful.

Suppositories for rectal administration of the compounds discussedherein can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di-, ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompounds of this invention are ordinarily combined with one or moreadjuvants appropriate to the indicated route of administration. Ifadministered per os, the compounds can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills can additionallybe prepared with enteric coatings.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions and suspensions can beprepared from sterile powders or granules having one or more of thecarriers or diluents mentioned for use in the formulations for oraladministration. The compounds can be dissolved in water, polyethyleneglycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil,sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.Other adjuvants and modes of administration are well and widely known inthe pharmaceutical art.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of active ingredient that can be combined with the carriermaterials to produce a single dosage form will vary depending upon thepatient and the particular mode of administration.

Doses/Quantities of Peptide Complexes. The amount of complex comprisinga drug or other pharmacologically active agent for administration to apatient to treat or prevent a disease condition will vary with the typeof drug, and will comprise a therapeutically effective amount thereof.Drug dosages for treating various conditions are well known in the art.Note in this regard, for example, Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 1996, Ninth Edition, McGraw-Hill, New York.

Routes of Administration. The complexes of the present invention can beadministered by a variety of methods, including, for example, orally,enterally, mucosally, percutaneously, or parenterally. Parenteraladministration is preferred, especially by intravenous, intramuscular,subcutaneous, intracutaneous, intraarticular, intrathecal, andintraperitoneal infusion or injection, including continuous infusions orintermittent infusions with pumps available to those skilled in the art.Alternatively, the complexes can be administered by means ofmicro-encapsulated preparations, for example those based on liposomes asdescribed in European Patent Application 0 213 523.

Treatment Regimens and Methods of Treatment. The regimen for treating apatient with the compounds and/or compositions of the present inventionis selected in accordance with a variety of factors, including the age,weight, sex, diet, and medical condition of the patient, the severity ofthe condition, the route of administration, pharmacologicalconsiderations such as the activity, efficacy, pharmacokinetic, andtoxicology profiles of the particular pharmacologically active compoundsemployed.

Administration of the drug complexes disclosed herein should generallybe continued over a period of several days, weeks, months, or years.Patients undergoing treatment with the drug complexes disclosed hereincan be routinely monitored to determine the effectiveness of therapy forthe particular disease or condition in question.

Continuous analysis of the data obtained by these methods permitsmodification of the treatment regimen during therapy so that optimalamounts of the pharmacologically active substance in the peptide complexare administered, and so that the duration of treatment can bedetermined as well. Thus, the treatment regimen/dosing schedule can berationally modified over the course of therapy so that the lowestamounts of drug compound is administered, and so that administration ofsuch compounds is continued only so long as is necessary to successfullytreat the disease or condition. In general, the dosage may be from about0.001, 0.01, 0.05, 0.1 or 0.5 to about 10, 50, 100, 500 or 1000 mg/kg,with dosages of about 0.05 to about 500 mg/kg preferred, and dosages ofabout 0.5 to about 5 mg/kg most preferred, depending upon the particulardisease, condition of the subject, route of administration, compoundformulation, etc.

Disorders or diseases that may be treated by the methods of the presentinvention are, in general, mitochondrial disorders, particularly nuclearand mitochondrial genome disorders such as Friedreich's ataxia, humanmitochondrial trifunctional protein deficiency, sudden infant deathsyndrome, Kearns-Sayre syndrome, and Leber's Hereditary OpticNeuropathy.

In some embodiments, useful for the diagnosis of mitochondrial diseasesin mammalian, particularly human, subjects, the compound of interest isa detectable group. The conjugate containing the detectable group can becontacted to cells or tissues, typically mammalian cells or tissues suchas muscle cells or tissues, by any suitable technique in vivo or invitro, for example by techniques conventional in the field of histology.The cells or tissues can be mounted, washed, and examined for thepresence of the detectable group, e.g., by microscopy, withmitochondrial disease being indicated by an abnormal increase, abnormaldecrease, abnormal distribution, or abnormal formation of mitochondria(e.g., accumulation of excessive mitochondrial in the subsarcolemmaregion) as compared to cells or tissues from a subject free ofmitochondrial disease. Note that mitochondria can currently be detectedonly using histologic stains and techniques. Thus, only fixed tissuescan be examined by light microscope or by electron microscope formitochondrial defects such as Leber's Hereditary Optic Neuropathy.However, using the strategy of attaching a bioactive signaling compound,such as a metal ion (boron, for example) or a radiolabeled fatty acid(to be detected by PET scanning) to the compound or conjugate of theinvention (e.g., TAT-mMDH-peptide) sequence, would allow investigationof active mitochondrial function in the living tissues. This is usefulfor following or monitoring recovery of tissues after disease, and allowbetter treatment of human disease. Also, the TAT-mMDH-peptide can beused to attach biotinylated groups which can then be localized withinthe mitochondria in living tissues. This is of great use when examininghistological samples because it clearly identifies mitochondria that arehealthy at the time of tissue harvest and fixation. Thus, theapplication and potential use of compounds or conjugates of theinvention to detect active mitochondrial function in the living tissuesand organs is great and will contribute to evaluation and management ofpatients in health and disease.

The present invention is explained in greater detail in the followingnon-limiting examples.

Example 1 Trifunctional Protein Conjugates for the Treatment ofMitochondrial Trifunctional Protein Deficiency

A. TAT-mMDH-eGFP

A fusion protein was constructed using the TAT region from the HIVvirus, a mitochondrial targeting leader peptide, and a reporter protein(green fluorescent protein, GFP) (FIG. 2A) (S. Schwarze, et al., Science285:1569-1572 (1999)). A second fusion protein was constructed thatincluded a mitochondrial targeting sequence (MTS), mitochondrial malatedehydrogenase (mMDH), within the TAT-fusion protein in addition to themitochondrial targeting leader peptide and reporter protein. Thetransduction of TAT-mMDH-eGFP was compared to that of TAT-GFP (aTAT-fusion protein that does not contain a MTS) into isolatedmitochondria, NIH 3T3, and differentiated PC12 cells in culture, andmultiple tissues of adult and pregnant mice. These proteins transversedboth the cell membrane and mitochondrial membranes, and crossed theplacenta into the fetus as well. While the mechanism of the proteinmovement across cell membranes is not understood, it is clear thatTAT-fusion proteins get into mitochondria because of the TAT-peptide andnot because of the MTS. Furthermore, when a MTS is present(TAT-mMDH-eGFP) the fusion protein is retained within the mitochondriaover time because of processing of the MTS by mitochondrial proteasemechanisms. This is an important finding since the TAT-fusion proteinfollows a chemical gradient and easily transduces back out of themitochondria or cell if it is not localized by processing or formationof a larger protein complex.

Recognition of the mMDH signal sequence and transduction of membranes inisolated mitochondria: To show that the mMDH signal peptide wasrecognized by the mitochondrial processing peptidases in the matrix,fresh rat liver mitochondria were isolated and shattered via sonication.The matrix contents were recovered and incubated with purifiedTAT-mMDH-eGFP (P. Grant, et al., Nucleic Acids Research 14:6053-6066(1986); E. Sztul, et al., Journal of Biological Chemistry263:12085-12091 (1988)). The matrix signal peptidase progressivelycleaved the mMDH signal sequence at two sites yielding two cleavageproducts of an estimated molecular mass of 32 kDa and 28.9 kDa (FIG. 2,A-C). FIG. 2D shows the resultant three bands representing the fulllength and the two cleavage products of the purified protein at theexpected molecular masses.

To show that the TAT sequence can cross mitochondrial membranes,respiring mitochondria were isolated from rat liver and incubated withpurified TAT-GFP and TAT-mMDH-eGFP protein for 15 minutes. Allmitochondrial incubation reactions were then treated with Proteinase Kto digest proteins that had not entered the mitochondrial matrix toensure that the proteins on the SDS-PAGE, or western blot, onlyconsisted of proteins inside mitochondria. Both TAT-GFP andTAT-mMDH-eGFP were found inside mitochondria as detected by anti-GFPantibody (FIG. 2E). Interestingly, TAT-mMDH-eGFP did not show processing(smaller MW on SDS-PAGE) after transduction into mitochondria. Oneexplanation for this may be that the transduced protein is rapidlycrossing the outer membrane and entering the intermembrane space but ismuch slower to cross the inner membrane into the matrix where theprocessing peptidases are located. TAT-fusion proteins may cross theinner membrane more slowly due to the higher protein content of theinner membrane.

TAT fusion proteins cross cell membranes: TAT-mMDH-eGFP and TAT-GFPfusion proteins transduced efficiently into cultured cells as shown byincubating purified protein with cultured NIH 3T3 cells. GFP activitywas detected using confocal microscopy. In cells incubated with eitherTAT-GFP or TAT-mMDH-eGFP the signal was spread throughout the cells,although not within the nucleus, and at the same intensity for bothfusion proteins (FIG. 3). As controls, cells were also incubated withrecombinant GFP and mMDH-eGFP, both without a TAT sequence. No GFPsignal was detectable within cells incubated with either protein (datanot shown) demonstrating that the GFP signal found within cells andmitochondria is not due to endocytic uptake or simple diffusion butrather the presence of the TAT sequence.

TAT fusion proteins cross mitochondrial membranes in cultured cells: NIH3T3 cells were co-incubated with purified fusion protein andCMX-Rosamine, a mitochondria-specific dye that is sequestered andfluoresces in mitochondria (N. Tarasova, et al., J. Biol. Chem.272:14817-14824 (1997); M. Yasuda, et al., J. Biol. Chem.273:12415-12421 (1998); H. Wang, et al., Cell 87:629-638 (1996)), inorder to demonstrate the presence of the GFP protein in mitochondria ofcultured cells. The cells were viewed using a confocal microscope withtwo lasers set at different wavelengths; 488λ to detect the GFP signalfrom the TAT fusion protein and 543λ to detect the CMX-Rosamine signalfrom mitochondria. The images were superimposed to see overlapping areasof fluorescence. Both TAT-GFP and TAT-mMDH-eGFP were present inmitochondria shortly after exposure to the TAT-fusion proteins as shownby the yellow color from the overlapping images (FIG. 3).

GFP signal persists over time in TAT-mMDH-eGFP treated cells: TAT fusionproteins are reported to be dependent on concentration gradients (S.Schwarze, et al., Science 285:1569-1572 (1999)). Removal of excessfusion protein from the surrounding media causes the TAT to diffuse outof the cells and their organelles. Purified fusion protein was incubatedwith cells to demonstrate that when the mMDH signal sequence isrecognized and cleaved in the TAT-mMDH-eGFP construct, the TAT diffusesout of the mitochondria leaving behind the eGFP. Cultured NIH 3T3 orPC12 cells were incubated with purified fusion protein for 1 hour, afterwhich the cells were washed, fresh medium added, and the cells placedback into the incubator for 1, 24, 48, or 72 hours. The cells were thenincubated with CMX-Rosamine, and the images from both lasers overlappedas described above. Little to no GFP signal was detected in cells ormitochondria of TAT-GFP treated cells (FIG. 3). However, the GFP signalin TAT-mMDH-eGFP treated cells persisted up to 72 hours after initialincubation (FIG. 3). The GFP signal in these cells was primarily withinmitochondria regardless of cell type.

The time course of TAT-GFP transduction into PC12 cells confirmed thatthis is a relatively fast process with signal being visible within 3minutes of application (FIG. 4). Signal continues to increase over 20minutes. The decreased rate of fluorescence change after 20 min may beaccounted for by the time needed for TAT to organize and create a poreto allow efficient movement across the cell membrane.

GFP signal found in liver and heart mitochondria of mice injected withTAT-mMDH-eGFP: Mice were injected intraperitoneal (ip) with 2 mg/kg ofeither TAT-GFP or TAT-mMDH-eGFP and sacrificed either 17 hours later, or5 days later. Liver, heart, kidney, and brain tissues were sectioned andexamined under the confocal microscope (FIGS. 5 and 7). In addition,mitochondria and cytosolic fractions were isolated from the livers ofthe 5 day animals and separated by SDS-PAGE (25 μg total protein perlane) with transfer to nitrocellulose for staining with anti-GFPantibody (Molecular Probes) (FIG. 6). A strong GFP signal was detectablein heart and liver of TAT-mMDH-eGFP injected mice as detected at 17hours (FIG. 5 A-C). GFP signal was present for the TAT-GFP mice (FIG. 5,D-E) though it was qualitatively not as strong as the TAT-mMDH-eGFPgroup. At 5 days after injection, however, the differences were muchgreater. Heart, and to a lesser extent, liver and brain, from animalsinjected with TAT-mMDH-eGFP had very strong signal (FIG. 5, G-I).Kidney, in particular, had a very strong signal after transduction ofTAT-mMDH-eGFP at 5 days (FIG. 7 D). In contrast, the animals injectedwith TAT-GFP had little signal visible in liver or brain, and moderatesignal in heart (FIG. 5). There was almost no GFP signal in kidney fromTAT-GFP at 5 days (FIG. 7E). FIG. 6 demonstrates that the entireTAT-mMDH-GFP signal in the 5 day animals is within the mitochondria andthe transduced protein has been processed (smaller molecular weight). Inthe 5 day animals injected with TAT-GFP, all of the GFP is found in thecytosol with no GFP staining in mitochondria. These in vivo results areconsistent with the in vitro cell culture results and show that cleavageof the mMDH signal sequence of TAT-mMDH-eGFP localized the eGFP proteinwithin the mitochondrial matrix. The TAT-GFP was not cleaved anddiffused out of the mitochondria, and out of the tissues.

TAT-fusion proteins cross the placenta. Pregnant mice at 18-19 days ofgestation were injected intraperitoneally (ip) with 2 mg/kg ofTAT-mMDH-eGFP. Sacrifice of the mother 24 hours later revealed extremelystrong GFP in heart and served as a positive control (FIG. 7A). Thefetus showed moderate GFP signal in heart at 24 hours after maternalinjection indicating that the TAT-fusion protein easily crossed theplacenta and entered the fetal circulation (FIG. 7B). A very strong GFPsignal was present in heart from the 24 hour old pup indicating thatTAT-mMDH-GFP had been processed and GFP remained within the mitochondriaover time (FIG. 7C). Interestingly, the red blood cells (RBC's) wereheavily stained in all tissues examined (arrows in FIG. 7C) suggestingthat TAT-fusion proteins may concentrate in these cells. Pups from thisexperiment continued to show strong GFP fluorescence in heart and liveras far out as 7 days after maternal injection of TAT-mMDH-eGFP (data notshown). This suggests that the transduced protein may have a relativelylong half-life in the mitochondria if the TAT peptide is removed.

The intensity and persistence of the GFP signal in TAT-mMDH-eGFPinjected mice, as compared to TAT-GFP mice, correlated well with thepersistence of GFP in the cell culture data. The strong GFP signal inTAT-mMDH-eGFP mice 5 days after injection is due to cleavage of the mMDHsignal sequence within the mitochondria, otherwise it would not persist(FIGS. 5, 6, 7). Therefore, the intensity and persistence of the GFPsignal, and processing of the MTS, signifies it is within mitochondria.These results illustrate that not only is this fusion protein able todeliver and localize a protein to mitochondria in vitro, but in vivo aswell. Furthermore, these results show that the present invention, usingTAT combined with a mitochondrial signal sequence is a feasible way totarget and localize exogenous compounds to mitochondria.

Transduction of TAT-fusion proteins across the cell membrane causesphosphatidylserine flip. Transduction of TAT-fusion proteins across thecell membrane causes membrane phospholipids rearrangement as evidencedby a flip in the phosphatidylserine (PS) from inner to outer plasmamembrane. TAT interacts with the negative charges on the phospholipidsmembrane to transduce. The PS flip is detected by Annexin-V staining incell culture. Cells treated for 12 hours with 300 μM H₂O₂ stainpositively with Annexin-V (FIG. 11B, while untreated cells do not haveAnnexin-V signal (FIG. 11C. Similarly, cells incubated with 0.01 mg.mlTAT-GFP also stain with Annexin V (FIG. 11D,E. However, pre-treatment ofcells with polylysine prior to incubation with TAT-GFP results in noAnnexin-V signal nor is any TAT-GFP signal detected in the cell (FIG.11F) In FIG. 11G fluorescent levels of activated Caspase-3 from culturedcells are shown after treatment with either 0.01 mg/ml (29.5 μM) TAT-GFP(diagonal bars) or hydrogen peroxide for 12 hours (solid black bars).Apoptosis is not initiated by this membrane rearrangement either in vivoor in vitro and thus, cell death is not expected in vivo.

B. Mitochondrial Trifunctional Protein Deficiency

TAT-fusion proteins offer a method of delivering biologically activeproteins to correct or repair mitochondrial defects. However, tworequirements must be met in order for this to occur: 1) The fusionprotein must target only the mitochondria and remain located there.Non-specific targeting of a protein to a different organelle must beavoided because it may disrupt a vital process. 2) The fusion proteinmust achieve biological activity inside the mitochondria. The presentinvention meets both of these requirements.

Animal Model of Trifunctional Protein deficiency: TFPα deficient miceare maintained in the heterozygous state because the homozygous state islethal to the neonatal animal. Specifically, the homozygous TFPα^(−/−)animals develop fatty liver (lipidosis) quickly after birth, withdegenerative changes in cardiac and diaphragmatic myocytes somewhatlater. Affected homozygous animals die within 36 hours of birth.

Tissue culture and Isolation of Primary Hepatocytes: The pregnant mouseis killed by cervical dislocation at 19 days of gestation and theembryos removed using sterile technique. The embryos are washed inphosphate buffered saline (PBS) with antibiotics, transferred to a Petridish with fresh PBS, and a piece of tail is cut for genotyping. Theliver is dissected out, washed in PBS with antibiotics, minced andwashed in PBS. The PBS is replaced with fresh PBS and antibioticscontaining 0.25% trypsin (1 ml for 100 mg of tissue) and incubated at 4°C. for 6 to 18 hrs. The supernatant is then removed; the remainingtissue incubated at 36.5° C. for 30 minutes, and warmed tissue media(Ham's F12) with 10% fetal calf serum is added with gentle pipetting ofthe tissue until the cells are dispersed. Cell viability is determinedusing trypan blue and cells are plated at 1×10⁶ cells per ml.

Primary and stable cell lines in culture are maintained in a 5% CO₂atmosphere at 37° C. with media appropriate for the cell type, such asDulbecco's Modification of Eagle's Medium (DMEM) with 5% bovine serumfor NIH 3T3 cells. For transduction experiments with TAT-fusionproteins, the culture media is replaced with PBS for 15 minutes duringwhich time the transduced protein is applied to the cells, the cells arethen washed with PBS and the media is replaced.

TAT-fusion protein construction and purification: The cDNA to beexpressed is subcloned in-frame into the vector containing the TAT aminoacid sequence (FIG. 8) and when expressed has the TAT sequence at theN-terminus along with a 6×His tag for purification on a nickel affinity(Ni-NTA) column. The purification strategy for these recombinantproteins includes a denaturing step with urea and shock denaturing on anion exchange column following published methods (M. Becker-Hapak, etal., Methods (Duluth) 24:247-256 (2001)). The finished protein isquantified, sterile filtered, and frozen at −70° C. with 10% glycerol.The cDNA for mouse TFPα is cloned into the cloning site of the pTATvector and, after sequencing the cDNA construct, the fusion protein isoverexpressed in bacteria.

Isolation of mitochondria: Intact, respiring mitochondria are preparedfrom heart using limited tissue digestion with Nagarse (0.4 mg/ml)followed by tissue disruption with a Polytron at medium speed. Themitochondria are then isolated by differential centrifugation. Use ofNagarase results in recovery of virtually all intact mitochondria (D.Rickwood, et al., In Mitochondria. A Practical Approach. V. M.Darley-Usmar, Rickwood, D., and Wilson, M. T., editors. IRL PressLimited, Oxford, England. 1-16 (1987); E. Lesnefsky, et al., AmericanJournal of Physiology 273: H1544-H1554 (1997)). Mitochondria isolatedfrom rat liver following protocols well-known in the art, are used toensure that all assays are working (D. Rickwood, et al., InMitochondria. A Practical Approach. V. M. Darley-Usmar, Rickwood, D.,and Wilson, M. T., editors. IRL Press Limited, Oxford, England. 1-16(1987)).

Histology: Electron microscopy (EM) is used to examine cellultrastructure and mitochondrial morphology ex vivo of liver and heartfrom treated, control, and untreated knock-out animals. Tissue isisolated from heart and liver and resuspended in fixative (4%glutaraldehyde, 100 mM sucrose, and 100 mM cacodylate buffer, pH 7.4),dehydration, and embedding for sectioning.

Tissue histology at the light microscope level is also used to evaluatehepatic, cardiac, and skeletal muscle responses to TAT-TFPα fusionprotein rescue in the homozygous animal, and possible scar formationfrom defects in fatty acid oxidation in long-term follow-up (J.Fruchart, et al., Current Opinion In Lipidology 10:245-257 (1999)).Hematoxylin and eosin staining of cross sections from treated andcontrol hearts and livers-provide data on phenotypic changes and tissuelipidosis. Oil Red O-stain is used on frozen sections to evaluate micro-and macrovesicular fatty infiltration. In addition, trichrome staining,or Azan-Mallory staining (K. Watanabe, et al., Journal of BiologicalChemistry 275:22293-22299 (2000)) is used to determine the amount ofscar tissue formation on long-term follow-up (4 weeks) after treatment.Images are scanned and digitized and the volume of scar tissuequantified.

Assessment of mitochondrial respiration: In order to follow the impactof fatty oxidation defects on mitochondrial injury. Mitochondrialrespiration ratios will be determined using total mitochondria isolatedfrom homozygous treated and untreated hearts and livers, and oxygenconsumption measured with an oxygraph and Clark oxygen electrode (YellowSpring Instruments, Yellow Spring, Ohio) (D. Rickwood, et al., InMitochondria. A Practical Approach. V. M. Darley-Usmar, Rickwood, D.,and Wilson, M. T., editors. IRL Press Limited, Oxford, England. 1-16(1987)). Cultured myocytes are evaluated similarly (Z. Khuchua, et al.,Journal of Biological Chemistry 273:22990-22996 (1998)). Oxygenconsumption by heart mitochondria is measured in air saturated medium ina closed container with a magnetic stir bar at 28° C. A volume of 100 μLof the mitochondrial suspension is added to the respiration buffer whichcontains 0.3 M sucrose, 1 mM EGTA, 5 mM Mops, 5 mM KH₂PO₄, and 0.1% BSAat pH 7.4. Basal respiration (V_(o)) is recorded for 3 minutes, pyruvateand malate are added (final concentrations 5 mM and 2.5 mM,respectively), and state 3 respiration is stimulated by 60 μM ADP tosubmaximal level (V_(ADP)). State 4 respiration is measured after theADP is depleted. Atractyloside, an inhibitor of the ATP-ADPtranslocator, is used to show this oxygen consumption is due tomitochondrial respiration. Oxygen consumption in rat liver mitochondriaprovides a second control to check the integrity of the system.

Determination of protein oxidation: —Reactive oxygen species attackamino acid residues in proteins to produce carbonyl functional groups.Carbonyl formation is used as a marker for protein oxidation (P. Evans,et al., Methods in Enzymology 300:145-156 (1999); A. Reznick, and L.Packer, Methods in Enzymology 233:357-363 (1994)) using2,4-dinitrophenylhydrazine (DNPH), which reacts with carbonyl groups toform protein hydrazones that are measurable spectrophotometrically. Byobtaining the spectra at 355-390 nm, the concentration of hydrazones/mlcan be calculated using Beer's Law: C=A/ε, where A in the absorption andε is the extinction coefficient. For DNPH, ε=45.45 nmol/ml. Levels ofmanganese superoxide dismutase (MnSOD) mRNA are measured by slot blotanalysis (Northern Blotting) using a cDNA probe for mouse MnSOD (Z.Chen, et al., Journal of Molecular & Cellular Cardiology 30:2281-2289(1998)).

Biochemical Assays: Blood from animals for analysis is obtained bycardiac puncture at the time of sacrifice. Glucose, urea and bilirubin,alanine aminotransferase (ALT), and gamma glutamyl transferase (GGT) ismeasured by automated clinical analyzer on less than 1 cc of wholeblood. Serum and tissue free fatty acids, serum acyl carnitine, andurine organic acids are analyzed using gas chromatography-massspectrometry (R. Boles, et al., Human Pathology 25:735-741 (1994)). Theactivities of LCHAD and long chain 3-ketoacyl-CoA thiolase are measuredon crude tissue extracts from heart and liver (R. Wanders, et al.,Biochemical & Biophysical Research Communications 188:1139-1145 (1992)).Since the active site of these two enzymes are located in the α and βsubunits of the TFP, a loss of activity in both is always associatedwith loss of the long chain 2,3 enoyl-CoA hydratase (TFPα chain). Gaschromatography-mass spectrometry is used to measure 3-hydroxy fattyacids in the cell culture media of TFPα deficient cells (P. Jones, etal., Clinical Chemistry 47:1190-1194 (2001)).

Positron Emission Tomography Scanning: Positron Emission Tomography(PET) Scanning is used to image cardiac and hepatic metabolism in TFPαdeficient mice. Monitoring glucose and/or fatty acid uptake by themyocardium and liver in these animals using microPET allows evaluationand prediction of hepatic and myocardial fatty acid oxidation aftertreatment with TAT-fusion proteins (S. Bergmann, et al., Journal ofInherited Metabolic Disease 24:657-674 (2001)).

Animals are sedated with halothane and maintained on 1% halothane. Theanimals are monitored to ensure that they remain sedated for the lengthof the procedure and PET scan. At the conclusion of the PET study theanimals are returned to their cages. PET imaging studies are performedprior to treatment with TAT-fusion proteins and redone after 1 week ofTAT-fusion protein treatment to detect changes in fatty acid oxidation.The anesthetized animal are injected with 2.0 mCi of the radiotracer(1-¹¹C palmitate and 1-¹¹C acetate) (S. Bergmann, et al., Journal ofInherited Metabolic Disease 24:657-674 (2001); S. Bergmann, et al.,Journal of Nuclear Medicine 37:1723-1730 (1996)) via the tail vein.Palmitate is used to estimate long chain fatty acid consumption bymitochondria, and acetate estimates oxygen consumption by mitochondria(V. Davila-Roman, et al., J. Am. Coll. Cardiol. 40:271-277 (2002)).Immediately after injection the animals are placed in the microPETgantry, positioned via anterior and lateral lasers and imaged for 20minutes over the torso to scan the heart and liver. Control mice arefasted for 12 hr. If there are difficulties injecting the radiotracerinto the tail vein, the right internal jugular vein may be used forinjection under direct visualization.

PET scans are performed on a Concorde Microsystems, Inc., microPET P4PET scanner. This device has no septa and therefore operates exclusivelyin three-dimensional imaging mode with axial and transaxial fields ofview of approximately 8 and 20 centimeters respectively. Thereconstructed resolution is approximately two millimeters in all threeaxes. Data are reconstructed using three-dimensional filteredbackprojection with a ramp filter cutoff at the Nyquist frequency (0.222mm⁻¹).

All image processing following reconstruction uses region-of-interest(ROI) analysis. A spherical ROI is placed over the anatomical region ofinterest. The maximum and average value in the PET scan of thethresholded pixels within the ROI is recorded. Palmitate and acetateutilization are expressed using the differential uptake ratio (DUR)method (K. Kubota, et al., Journal of Nuclear Medicine 37:1713-1717(1996)):

${DUR} = \frac{{counts}\text{/}{pixel}}{{Injection}\mspace{14mu} {dose}\text{/}{rat}{\mspace{11mu} \;}{body}\mspace{14mu} {weight}}$

Statistical Analysis: All values are presented as means ±SD. Forcomparisons of responses between different groups, unpaired t-tests oranalysis of variance are used. If the F-crit value is significant, thena pair-wise test (Student-Newman-Kuels) is performed. A p<0.05 isrequired in order to be significant.

The TAT peptide delivers an active mitochondrial protein in vitro. Amitochondrial targeting sequence is required for the matrix processingpeptidases to recognize and cleave thereby fixing the protein in thematrix space. Without the targeting sequence, the TAT sequence remainsattached and can facilitate diffusion of the TAT-fusion protein out ofthe matrix space, or inhibit normal folding and incorporation of thetransduced protein into a biologically active complex. Prior to thepresent invention, this has not been accomplished for matrix proteins.

Protocol: The cDNA containing the coding sequence of mouse TFPα (GenBankAccession #XM131963) is subcloned into the TAT-vector. Prior toexpression in E. coli the cDNA construct is sequenced to ensure that itis in frame. The A hemagglutinin (HA) tag is included for antibodydetection. Nickel affinity chromatography is used for purification ofthe protein, and low-pressure chromatography for denaturation. TAT-TFPαprotein purity and quantity are determined by gel electrophoresis.

The TAT-TFPα fusion protein is tested in cell culture to ensure ittargets to the correct space in mitochondria and is proteolyticallyprocessed. This is done utilizing both antibody to TFPα and HA, and byfluorescent labeling (Molecular Probes) of synthetic protein prior toincubation with cells in culture. Processed protein is also isolated foruse in sequencing by tandem MS-MS. The protein and mRNA levels of TFPβare determined using antibodies and cDNA probes specific for this geneproduct (J. Ibdah, et al., Journal of Clinical Investigation107:1403-1409 (2001)). Mitochondria are stained with CMX-H₂-Ros to checkfor integrity and co-localization with TFPα. The nuclei arecounter-stained with Hoechst 33342 fluorescent marker to identifynuclear membranes. Initially NIH 3T3 cells are used because of theirease of manipulation and the high number of mitochondria that can berecovered from these cells. The fate of the TAT-HA-MTS when cleaved fromthe precursor TAT-TFPα fusion protein is determined by following itslocation and integrity using antibodies against the HA portion. Thehistology of the transduced cells will be examined at the light and EMlevels, and mitochondrial respiration be measured on cells in culture aswe have done for cultured myocytes (Z. Khuchua, et al., Journal ofBiological Chemistry 273:22990-22996 (1998)). The appropriate controlsinclude TFPα without the TAT sequence, and TAT-TFPα without the MTS(TAT-TFPα^(−MTS)). The use of TAT-mMDH-eGFP in parallel experimentsensures that the mitochondria have been targeted.

Rescue of the phenotype of an animal transgenic for the loss of TFPfunction. Biological activity of TAT-fusion proteins has been shown invivo. However, until the present invention, transduction of biologicallyactive fusion proteins into the mitochondrial matrix has not been shown.This is a crucial step in the repair of mitochondrial defects leading tohuman disease. This invention shows that the TAT-TFPα fusion protein canlocalize to the inner membrane of the mitochondrial matrix and integrateinto a hetero-octamer of four α-subunits, and four β-subunits, to forman active TFP complex in vivo. In addition, since TFPβ is not stable andis rapidly degraded without TFPα, this invention shows that once theTAT-TFPα peptide is transduced into the mitochondria of a TFPα^(−/−)mouse and processed then TFPβ is detectable.

Protocol: The effects of transduction of the TAT-TFPα fusion protein isshown in three experimental groups:

-   -   1. Control Group—wild type C57BL/6J mice at 3 to 4 months of        age, as well as neonatal mice of the same strain. These animals        express normal levels of TFP.    -   2. Neonatal TFPα^(−/−) mice—neonatal mice homozygous for        ablation of the TFPα gene. This is most easily determined by        phenotype but is also be confirmed by Southern Blotting or PCR        analysis (64).    -   3. Pregnant heterozygous females (TFPα^(+/−))—pregnant dams        heterozygous for the TFPα gene, bred with males heterozygous for        the TFPα knockout state. Thus, 25% of the embryos will be        homozygous TFPα^(−/−) (knockout), 50% will be heterozygous        TFPα^(+/−), and 25% should be homozygous TFPα^(+/+).

Following injection of the TAT-TFPα fusion protein into mice, properdelivery to the mitochondria in heart, liver, brain, and skeletal muscleis assayed for based on its biochemical activity, immunolocalization tomitochondria, and total protein mass. Impact of the transduced proteinon the animal's growth and viability, and on tissue histology isdetermined using the above outlined protocol. TFP enzyme activity andother biochemical parameters are determined using the above outlinedprotocol. Positron Emission Tomography (micro-PET Scan) using[1-¹¹C]-palmitate and [1-¹¹C]-acetate as tracers is used to show thatthe rescued phenotype is able to utilize long-chain fatty acids forfuel.

The appropriate dosage and dosing intervals was determined using serialbiochemical assays, TFPα and TFPβ protein mass, and physical conditionin the homozygous TFPα^(−/−) animals. This information was correlatedwith microPET scans of homozygous animals using labeled long chain fattyacids as tracers and compared with microPET scans from normal controls.Thus, yielding a non-invasive method for long-term tracking ofmitochondrial function and functional recovery of TFP activity in theanimal.

Distribution of the TAT-TFPα fusion protein in multiple tissues is shownby immunohistochemistry and Western blotting.

The immune response to chronic administration of TAT-Fusion proteins isstudied to determine side effects of long-term administration of theprotein. For this purpose enzyme-linked immunosorbent assays (ELISA) areperformed on serum from animals injected for 1, 2, 4 and 12 months withTAT-fusion proteins (116). Inflammatory response is also examine in thecontrol and homozygous knock-out animals for evidence of impairedmitochondrial respiration, and mitochondrial oxidative damage.

Example 2 Frataxin Conjugates for the Treatment of Freidreich's Ataxia

Defects in mitochondrial function are common in human health anddisease. Because mitochondria have only a small genome, ˜16 kb, theymust import most of the hundreds of proteins needed for their functionfrom nuclear-encoded genes. Defects in these imported proteins arefrequent causes of disease and metabolic disorders. Friedreich's Ataxia(FA) is one such disease and is the most common autosomal recessiveataxia. FA is caused by lack of the nuclear-encoded, mitochondriallytargeted protein, Frataxin. FA is caused by a large expansion of a GAAtriplet-repeat sequence in the first intron of the Frataxin gene leadingto decreased transcription of full-length transcripts (elongation isinhibited) (P. Patel and G. Isaya, Am. J. Hum. Genet. 69:15-24 (2001)).The result being that Frataxin protein is severely deficient leading toprogressive iron accumulation and dysfunction in mitochondria (O. Gakh,et al., Biochemistry. 41:6798-6804 (2002)). Patients with Friedreich'sAtaxia present in childhood years with ataxia and limb weakness withprogressive cardiomyopathy and motoneuron dysfunction and die in the4^(th) or 5^(th) decade. Effective therapy for this progressive disorderhas not been achieved.

Human Frataxin is translated as a ˜29 kDa precursor protein with a 55amino acid mitochondrial targeting sequence at the amino terminus. Theprecursor is processed in 2 steps to a smaller ˜18 kDa mature peptide inthe mitochondrial matrix that is then assembled into a homopolymer of ˜1MDa binding approximately 5 atoms of iron per molecule (P. Cavadini, etal., Hum. Mol. Genet. 11:217-227 (2002); P. Cavadini, et al., J. Biol.Chem. 275:41469-41475 (2000)).

Using the protein transduction domain, TAT, exogenous proteins can bedelivered to mitochondria. Furthermore, by using a mitochondrialtargeting sequence, the protein can be specifically targeted tomitochondria and processed leaving the mature protein within themitochondrial matrix.

TAT-Frataxin fusion protein construct targets mitochondria. The cDNA formouse Frataxin is subcloned in-frame behind the TAT peptide andexpressed in E. Coli. The fusion protein is purified by affinitychromatography and purity determined by PAGE. Proper targeting isdetermined by incubation of precursor TAT-Frataxin with mitochondria toshow proper processing and transduction into the matrix compartment.—Transduction across both cell and mitochondrial membranes withlocalization in mitochondria is shown by application of TAT-Frataxinfusion protein to cells in culture.

The TAT-Frataxin fusion protein has been shown to transduce into NIH-3T3cells (FIG. 12). NIH 3T3 cells were co-incubated with purified fusionprotein and CMX-Rosamine, a mitochondria-specific dye that issequestered and fluoresces in mitochondria (N. Tarasova, et al., J.Biol. Chem. 272:14817-14824 (1997); M. Yasuda, et al., J. Biol. Chem.273:12415-12421 (1998); H. Wang, et al., Cell 87:629-638 (1996)), inorder to demonstrate the presence of the TAT-Frataxin in mitochondria ofcultured cells (FIG. 12D). The cells were viewed using a confocalmicroscope and the images were superimposed to see overlapping areas offluorescence. Successful transduction and sequestering of the humanFrataxin into the mitochondria was observed.

The TAT-Frataxin Fusion Protein Rescues the FA Phenotype.

Animal Models of Frataxin Protein deficiency: The conditional knock-outmouse model of FA (FRDA deficiency) is available (H. Puccio, et al.,Nat. Genet. 27:181-186 (2001)). Both a cardiac-specific, and aneuronal-specific knock-out have been generated although there iscross-over in the phenotypes. The homozygous FRDA knock-out is embryoniclethal (M. Cossee, et al., Hum. Mol. Genet. 9:1219-1226 (2000)).

Tissue culture and Isolation of Primary Cells: Primary and stable celllines in culture are maintained in a 5% CO₂ atmosphere at 37° C. withmedia appropriate for the cell type, such as DMEM with 5% bovine serumfor NIH 3T3 cells. For transduction experiments with TAT-fusionproteins, the culture media is replaced with PBS for 15 minutes duringwhich time the transduced protein is applied to the cells, the cells arethen washed with PBS and the media is replaced. Primary cells(fibroblasts) from FA patients or the knock-out mice, are grown inestablished conditions and the TAT-FA fusion protein transduced asoutlined in the above protocols. Biochemical and histological protocolsare outlined below.

TAT-fusion protein construction and purification: The cDNA for mouseFrataxin (precursor form: GenBank accession U95736) is subclonedin-frame into the cloning site of the pTAT vector. The cDNA construct issequenced to ensued fidelity and that the Frataxin cDNA is in frame.Following sequencing the cDNA construct is overexpressed in bacteria.When expressed, the fusion protein has the TAT sequence at theN-terminus along with a 6×His tag for purification on a nickel affinity(Ni-NTA) column (FIG. 1 shows the strategy for construction ofTAT-mMDH-GFP). The purification strategy for these recombinant proteinsincludes a denaturing step with urea and shock denaturing on an ionexchange column following published methods (M. Becker-Hapak, et al.,Methods (Duluth) 24:247-256 (2001)). The purified protein is quantified,sterile filtered, and frozen at −70° C. with 10% glycerol. No mMDHtargeting sequence is required because Frataxin has its own MTS. Theadult and neonatal mice are injected in the peritoneum with 2 mg/kg bodyweight of the fusion protein. Cell culture media is prepared at aconcentration of 0.01 mg/ml fusion protein.

Isolation of mitochondria: Intact, respiring mitochondria are preparedfrom heart using limited tissue digestion with Nagarse (0.4 mg/ml)followed by tissue disruption with a Polytron at medium speed. Themitochondria are then isolated by differential centrifugation. Use ofNagarase results in recovery of virtually all intact mitochondria (D.Rickwood, et al., In Mitochondria. A Practical Approach. V. M.Darley-Usmar, Rickwood, D., and Wilson, M. T., editors. IRL PressLimited, Oxford, England. 1-16 (1987); E. Lesnefsky, et al., AmericanJournal of Physiology 273: H1544-H1554 (1997)). Mitochondria isolatedfrom rat liver following protocols well-known in the art, are used toensure that all assays are working (D. Rickwood, et al., InMitochondria. A Practical Approach. V. M. Darley-Usmar, Rickwood, D.,and Wilson, M. T., editors. IRL Press Limited, Oxford, England. 1-16(1987)).

Histology: Electron microscopy (EM) is used to examine cellultrastructure and mitochondrial morphology ex vivo of liver and heartfrom treated, control, and untreated knock-out animals. Tissue isisolated from heart and liver and resuspended in fixative (4%glutaraldehyde, 100 mM sucrose, and 100 mM cacodylate buffer, pH 7.4),dehydration, and embedding for sectioning.

Tissue histology at the light microscope level is used to evaluatehepatic, cardiac, and brain responses to TAT-Frataxin fusion proteinrescue in the transgenic animal. Phenotypic changes are followed usinghematoxylin and eosin staining of cross sections from treated andcontrol hearts and brains with liver serving as a control. In addition,trichrome staining, or Azan-Mallory staining (K. Watanabe, et al.,Journal of Biological Chemistry 275:22293-22299 (2000)) is used todetermine the amount of scar tissue formation in heart on long-termfollow-up (4 weeks) after treatment. Images are scanned and digitizedand the volume of scar tissue quantified. Immunohistology is performedon unstained sections to show presence of Frataxin protein in multipletissues.

Assessment of mitochondrial respiration: Mitochondrial respirationratios will be determined using total mitochondria isolated fromhomozygous treated and untreated hearts and livers, and oxygenconsumption measured with an oxygraph and Clark oxygen electrode (YellowSpring Instruments, Yellow Spring, Ohio) (D. Rickwood, et al., InMitochondria. A Practical Approach. V. M. Darley-Usmar, Rickwood, D.,and Wilson, M. T., editors. IRL Press Limited, Oxford, England. 1-16(1987)). Oxygen consumption by mitochondria is measured in air saturatedmedium in a closed container with a magnetic stir bar at 28° C.Atractyloside, an inhibitor of the ATP-ADP translocator, is used to showthis oxygen consumption is due to mitochondrial respiration. Rat livermitochondria are used as a second control to check the integrity of thesystem.

Determination of protein oxidation: Determination of protein oxidation:Reactive oxygen species attack amino acid residues in proteins toproduce carbonyl functional groups. The carbonyl formation will be usedas a marker for protein oxidation (P. Evans, et al., Methods inEnzymology 300:145-156 (1999); A. Reznick, and L. Packer, Methods inEnzymology 233:357-363 (1994)). 2,4-dinitrophenylhydrazine (DNPH) reactswith carbonyl groups to form protein hydrazones which are measurablespectrophotometrically. By obtaining the spectra at 355-390 nm, theconcentration of hydrazones/ml can be calculated using Beer's Law:C=A/ε, where A in the absorption and ε is the extinction coefficient.For DNPH, ε=45.45 nmol/ml. Levels of manganese superoxide dismutase(MnSOD) mRNA are measured by slot blot analysis (Northern Blotting)using a cDNA probe for mouse MnSOD (Z. Chen, et al., Journal ofMolecular & Cellular Cardiology 30:2281-2289 (1998)).

Phenotypic Assays: Animals are followed for the development ofcardiomyopathy using echocardiography and growth rate, and for signs ofneuromotor weakness by exercise in wheel cage and proprioception as inpublished data (H. Puccio, et al., Nat. Genet. 27:181-186 (2001)).Cardiac function is determined longitudinally by cardiac ultrasound (seeFIG. 2) and ex vivo by heart to body weight. The neuroanatomy of brainfrom animals with advanced disease is evaluated using histology, EM, andimmunohistochemistry.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-7. (canceled)
 8. A method of delivering a compound of interest intothe mitochondria of a cell, comprising: contacting a conjugate to saidcell, wherein said conjugate comprises: (a) a mitochondrialmembrane-permeant peptide; (b) a compound of interest; and (c) amitochondrial targeting sequence linking said mitochondrialmembrane-permeant peptide and said compound of interest, wherein saidtargeting sequence is cleaved within the mitochondrial matrix and notcleaved within the cellular cytoplasm of a cell into which saidconjugate is delivered; so that said compound of interest is deliveredinto the mitochondria within said cell. 9-16. (canceled)
 17. A method oftreating a mitochondrial disorder in a subject in need thereof,comprising administering a conjugate to said subject in an amounteffective to treat said mitochondrial disorder; wherein the conjugatecomprises: (a) a mitochondrial membrane-permeant peptide; (b) an activemitochondrial protein or peptide; and (c) a mitochondrial targetingsequence linking said mitochondrial membrane-permeant peptide and saidactive mitochondrial protein or peptide, wherein said targeting sequenceis cleaved within the mitochondrial matrix and not cleaved within thecellular cytoplasm of a cell into which said conjugate is delivered. 18.The method of claim 17, wherein said mitochondrial disorder is selectedfrom the group consisting of Friedreich's ataxia, human mitochondrialtrifunctional protein deficiency, sudden infant death syndrome,Kearns-Sayre syndrome and Leber's Hereditary Optic Neuropathy.
 19. Themethod of claim 8, wherein said cell is in vitro.
 20. The method ofclaim 8, wherein said cell is in vivo.
 21. The method of claim 8,wherein said mitochondrial membrane-permeant peptide is proteintransduction domain peptide.
 22. The method of claim 8, wherein saidmitochondrial membrane-permeant peptide is an HIV-TAT peptide.
 23. Themethod of claim 8, wherein said mitochondrial targeting sequence is amitochondrial malate dehydrogenase cleavage sequence.
 24. The method ofclaim 8, wherein said mitochondrial targeting sequence is amitochondrial processing peptide cleavage sequence.
 25. The method ofclaim 8, wherein said compound of interest is a detectable group. 26.The method of claim 8, wherein said compound of interest is an activemitochondrial protein or peptide
 27. The method of claim 26, whereinsaid active mitochondrial protein or peptide and said mitochondrialtargeting sequence are heterologous or homologous.
 28. The method ofclaim 26, wherein said active mitochondrial protein or peptide isfrataxin.
 29. The method of claim 26, wherein said active mitochondrialprotein or peptide is mitochondrial trifunctional protein alpha.
 30. Themethod of claim 26, wherein said mitochondrial targeting sequence is amitochondrial malate dehydrogenase cleavage sequence and said activemitochondrial protein or peptide is frataxin.
 31. The method of claim26, wherein said mitochondrial targeting sequence is a mitochondrialmalate dehydrogenase cleavage sequence and said active mitochondrialprotein or peptide is mitochondrial trifunctional protein alpha.
 32. Themethod of claim 26, wherein said mitochondrial targeting sequence is afrataxin cleavage sequence and said active mitochondrial protein orpeptide is frataxin.
 33. The method of claim 17, wherein saidmitochondrial membrane-permeant peptide is protein transduction domainpeptide.
 34. The method of claim 17, wherein said mitochondrialmembrane-permeant peptide is an HIV-TAT peptide.
 35. The method of claim17, wherein said mitochondrial targeting sequence is a mitochondrialmalate dehydrogenase cleavage sequence.
 36. The method of claim 17,wherein said mitochondrial targeting sequence is a mitochondrialprocessing peptide cleavage sequence.
 37. The method of claim 17,wherein said active mitochondrial protein or peptide and saidmitochondrial targeting sequence are heterologous or homologous.
 38. Themethod of claim 17, wherein said active mitochondrial protein or peptideis frataxin.
 39. The method of claim 17, wherein said activemitochondrial protein or peptide is mitochondrial trifunctional proteinalpha.
 40. The method of claim 17, wherein said mitochondrial targetingsequence is a mitochondrial malate dehydrogenase cleavage sequence andsaid active mitochondrial protein or peptide is frataxin.
 41. The methodof claim 17, wherein said mitochondrial targeting sequence is amitochondrial malate dehydrogenase cleavage sequence and said activemitochondrial protein or peptide is mitochondrial trifunctional proteinalpha.
 42. The method of claim 17, wherein said mitochondrial targetingsequence is a frataxin cleavage sequence and said active mitochondrialprotein or peptide is frataxin.